5-FU-miR-15a Inhibits Activation of Pancreatic Stellate Cells by Reducing YAP1 and BCL-2 Levels In Vitro

Chronic pancreatitis is characterized by chronic inflammation and fibrosis, processes heightened by activated pancreatic stellate cells (PSCs). Recent publications have demonstrated that miR-15a, which targets YAP1 and BCL-2, is significantly downregulated in patients with chronic pancreatitis compared to healthy controls. We have utilized a miRNA modification strategy to enhance the therapeutic efficacy of miR-15a by replacing uracil with 5-fluorouracil (5-FU). We demonstrated increased levels of YAP1 and BCL-2 (both targets of miR-15a) in pancreatic tissues obtained from Ptf1aCreERTM and Ptf1aCreERTM;LSL-KrasG12D mice after chronic pancreatitis induction as compared to controls. In vitro studies showed that delivery of 5-FU-miR-15a significantly decreased viability, proliferation, and migration of PSCs over six days compared to 5-FU, TGFβ1, control miR, and miR-15a. In addition, treatment of PSCs with 5-FU-miR-15a in the context of TGFβ1 treatment exerted a more substantial effect than TGFβ1 alone or when combined with other miRs. Conditioned medium obtained from PSC cells treated with 5-FU-miR-15a significantly inhibits the invasion of pancreatic cancer cells compared to controls. Importantly, we demonstrated that treatment with 5-FU-miR-15a reduced the levels of YAP1 and BCL-2 observed in PSCs. Our results strongly suggest that ectopic delivery of miR mimetics is a promising therapeutic approach for pancreatic fibrosis and that 5-FU-miR-15a shows specific promise.


Introduction
Chronic pancreatitis is associated with a 50% mortality rate and is one of the most substantial risk factors for pancreatic cancer development [1]. Chronic inflammation and fibrosis leads to structural damage, which impairs the organ's critical endocrine and exocrine functions [2,3]. Indeed, widespread fibrosis is the prominent histological feature of chronic pancreatitis and results from the progressive activation of pancreatic stellate cells (PSCs). The most common consequences of chronic pancreatitis includes recurrent or constant abdominal pain, diabetes mellitus (endocrine insufficiency), and maldigestion (exocrine insufficiency) [4]. Fundamentally, diagnosis of early-stage chronic pancreatitis is challenging as its features are shared with other disorders. Recent studies have shown that destroying pancreatic acinar cells induces a proinflammatory response that triggers PSCs activation, leading to widespread fibrosis [5].
Repeated pancreatic injury results in the dysregulation of pancreatic acinar cell function, which is typified by an increase in intracellular calcium, dysfunction of the endoplasmic reticulum and mitochondria, chronic activation of pancreatic enzymes, and alteration of lipid metabolic pathways [6][7][8]. These changes sequentially lead to impaired acinar cell autophagy. In addition, there is increased cell death, followed by immune cell infiltration

YAP1 and BCL-2 Are Upregulated, While miR-15a Is Downregulated during Chronic Pancreatitis
Recent publications demonstrate that miR-15a levels are downregulated in human chronic pancreatitis specimens compared to healthy controls [25]. To investigate the role of miR-15a in chronic pancreatitis, we first assessed miR-15a levels and its targets (YAP1 and BCL-2) upon the development of chronic pancreatitis in Ptf1aCre ERTM and Ptf1aCre ERTM ;LSL-KRAS G12D mice. These two animal models allow us to analyze different stages of pancreatitis progression. We implemented a four-week cerulein regimen that leads to the development of mild chronic pancreatitis [32]. Pancreatic histology in Ptf1a-Cre ERTM control mice were similar to those found in human chronic pancreatitis patients. This is characterized by inflammatory infiltrates, acinar cell atrophy, edema, and fibrosis. Notably, in Ptf1aCre ERTM ;LSL-Kras G12D mice, the same injury regimen leads to early neoplasia, characterized by extensive fibrosis and pancreatic exocrine insufficiency [33][34][35].
Immunohistochemical analysis of YAP1 and BCL-2 showed that both factors were upregulated in pancreatic tissues of Ptf1aCre ERTM and Ptf1aCre ERTM ;LSL-KRAS G12D mice treated with cerulein as compared to appropriate controls ( Figure 1A and B, respectively). Furthermore, the increase of both YAP1 and BCL-2 was observed in injured pancreatic acinar cells, early pancreatic neoplastic cells, and fibrotic microenvironment upon cerulein treatment ( Figure 1A,B). The overall increase in staining of YAP1 and BCL-2 was connected to the increase in the fibrotic component and injured pancreatic acinar cells that undergo acinar-to-ductal metaplasia toward early pancreatic neoplasia. treated with cerulein as compared to appropriate controls ( Figure 1A and 1B, res tively). Furthermore, the increase of both YAP1 and BCL-2 was observed in injured creatic acinar cells, early pancreatic neoplastic cells, and fibrotic microenvironment u cerulein treatment ( Figure 1A,B). The overall increase in staining of YAP1 and BCL-2 connected to the increase in the fibrotic component and injured pancreatic acinar cells undergo acinar-to-ductal metaplasia toward early pancreatic neoplasia.  Immunohistochemical staining of YAP1 and BCL-2 during the development of chronic pancreatitis. Pancreatic specimens were obtained from Ptf1aCre ERTM and Ptf1aCre ERTM ;LSL-Kras G12D mice were treated with tamoxifen for one week and subsequently with PBS (vehicle) and CER (cerulein) for four weeks and samples were collected 1, 2, 3, and 4 weeks after the injury. (A) Immunohistochemical stain of YAP1, and (B) immunohistochemical stain of BCL-2. The scale bar represents 100 µm. Black arrows point to fibrotic cells, black circles mark injured pancreatic acinar cells, and red arrows mark early pancreatic neoplastic cells positive for YAP1 or BCL-2 in Figure 1A and B, respectively. Furthermore, we found that miR-15a expression was significantly decreased in the pancreas of Ptf1aCre ERTM and Ptf1aCre ERTM ;LSL-KRAS G12D mice treated with cerulein as compared to the control (PBS-treated mice) (Figure 2A). In contrast, the levels of mRNA of Yap1 and Bcl2 were markedly increased in Ptf1aCre ERTM and Ptf1aCre ERTM ;LSL-KRAS G12D mice treated with cerulein as compared to the appropriate controls ( Figure 2B,C). Interestingly, compared with PBS-treated controls, the increase in Yap1 in the pancreas revealed a 6-fold and 14-fold upregulation in Ptf1aCre ERTM change in Ptf1aCre ERTM ;LSL-KRAS G12D mice under chronic pancreatitis conditions, respectively. Furthermore, we observed an increase in Bcl2 levels with a 3-fold and 4-fold upregulation in Ptf1aCre ERTM and Ptf1aCre ERTM ;LSL-KRAS G12 mice, respectively, as compared to appropriate controls, respectively. In addition, we assessed the activation of PSCs by determining the levels of Col1a1, Mmp9, and Il6 factors previously shown to be upregulated in PSCs during chronic pancreatitis [12,[36][37][38][39][40][41]. Our results demonstrate that the markers mentioned above are upregulated in Ptf1aCre ERTM and Ptf1aCre ERTM ;LSL-KRAS G12 treated with cerulein as compared to the controls (PBS-treated mice) ( Figure 2D-F). In addition, we assessed the protein levels of YAP1 and BCL-2 in Ptf1aCre ERTM ;LSL-KRAS G12D after four weeks of PBS or CER treatment. Our results showed that there is a significant increase in both proteins upon chronic injury ( Figure 2G).

pancreas of Ptf1aCre
and Ptf1aCre ;LSL-KRAS mice treated with cerulein as compared to the control (PBS-treated mice) (Figure 2A). In contrast, the levels of mRNA of Yap1 and Bcl2 were markedly increased in Ptf1aCre ERTM and Ptf1aCre ERTM ;LSL-KRAS G12D mice treated with cerulein as compared to the appropriate controls ( Figure 2B,C). Interestingly, compared with PBS-treated controls, the increase in Yap1 in the pancreas revealed a 6-fold and 14-fold upregulation in Ptf1aCre ERTM change in Ptf1aCre ERTM ;LSL-KRAS G12D mice under chronic pancreatitis conditions, respectively. Furthermore, we observed an increase in Bcl2 levels with a 3-fold and 4-fold upregulation in Ptf1aCre ERTM and Ptf1aCre ERTM ;LSL-KRAS G12 mice, respectively, as compared to appropriate controls, respectively. In addition, we assessed the activation of PSCs by determining the levels of Col1a1, Mmp9, and Il6 factors previously shown to be upregulated in PSCs during chronic pancreatitis [12,[36][37][38][39][40][41]. Our results demonstrate that the markers mentioned above are upregulated in Ptf1aCre ERTM and Ptf1aCre ERTM ;LSL-KRAS G12 treated with cerulein as compared to the controls (PBS-treated mice) ( Figure 2D-F). In addition, we assessed the protein levels of YAP1 and BCL-2 in Ptf1aCre ERTM ;LSL-KRAS G12D after four weeks of PBS or CER treatment. Our results showed that there is a significant increase in both proteins upon chronic injury ( Figure 2G).  Furthermore, we performed immunofluorescence staining of Ptf1aCre ERTM ;LSL-Kras G12D mice treated with tamoxifen for one week and subsequently with PBS or CER for four weeks with antibody against αSMA and Ki-67, and included representative images in Figure 3A. As shown, there are αSMA + cells that are positive for Ki-67, confirming the proliferative status of PSCs. In addition, we showed that there is a significant increase in fibrosis by Masson's Trichrome staining in CER-treated Ptf1aCre ERTM ;LSL-Kras G12D mice as compared to PBS-treated mice for four weeks ( Figure 3B).
PCR levels of Yap1. (C) Represents RT-PCR levels of Bcl2. (D) Represents RT-PCR levels of Col1a1. (E) Represent RT-PCR levels of Mmp9. (F) Represent RT-PCR levels of Il6. Data are represented as the mean ± SD; N = 3. *** p < 0.001 and **** p < 0.0001 by One-Way ANOVA. (G) Western blot analysis and quantification of YAP1 and BCL-2 levels in Ptf1aCre ERTM ;LSL-Kras G12D mice treated with tamoxifen for one week and subsequently with PBS (vehicle) or CER (cerulein) for four weeks. ACTIN was used as a loading control. Data are represented as the mean ± SD; N = 3. ** p < 0.01 using t-test. Furthermore, we performed immunofluorescence staining of Ptf1aCre ERTM ;LSL-Kras G12D mice treated with tamoxifen for one week and subsequently with PBS or CER for four weeks with antibody against αSMA and Ki-67, and included representative images in Figure 3A. As shown, there are αSMA + cells that are positive for Ki-67, confirming the proliferative status of PSCs. In addition, we showed that there is a significant increase in fibrosis by Masson's Trichrome staining in CER-treated Ptf1aCre ERTM ;LSL-Kras G12D mice as compared to PBS-treated mice for four weeks ( Figure 3B).

5-FU-miR-15a Significantly Reduced the Proliferation of Murine Pancreatic Stellate Cells In Vitro
Recent publications demonstrated that exogenous delivery of miR-15a and, specifically, 5-FU-miR-15a significantly reduced the expression levels of YAP1 and BCL2 in pancreatic and colorectal cancer cell lines, respectively, and led to growth inhibition [29,31]. Therefore, we performed a Cell-Titer Glo assay to assess the efficacy of 5-FU-miR-15a on the viability of murine pancreatic stellate cells. For six days, PSCs were treated with variable concentrations of TGFβ1, 5-FU, control (CTRL) miR, miR-15a, and 5-FU-miR-15a ( Figure 4). Our results showed that 5 ng/mL and 10 ng/mL TGFβ1 and 5-FU at 1 mM and 50 µM reduced the viability of PSCs. Importantly, 5-FU-miR-15a achieved the most potent inhibition of PSCs viability at nanomolar concentrations, while CTRL miR and miR-15a exert only a weak inhibitory effect.
stromal cells positive for Ki-67 + and pink arrows mark early pancreatic neoplastic cells positive for Ki-67 + in pancreas obtained from Ptf1aCre ERTM ;LSL-Kras G12D mice treated with CER, and (B) representative Masson's Trichrome staining with quantification. The scale bar represents 100 µm. Data are represented as the mean ± SD; N = 3. **** p < 0.0001 using t-test.

5-FU-miR-15a Significantly Reduced the Proliferation of Murine Pancreatic Stellate Cells In Vitro
Recent publications demonstrated that exogenous delivery of miR-15a and, specifically, 5-FU-miR-15a significantly reduced the expression levels of YAP1 and BCL2 in pancreatic and colorectal cancer cell lines, respectively, and led to growth inhibition [29,31]. Therefore, we performed a Cell-Titer Glo assay to assess the efficacy of 5-FU-miR-15a on the viability of murine pancreatic stellate cells. For six days, PSCs were treated with variable concentrations of TGFβ1, 5-FU, control (CTRL) miR, miR-15a, and 5-FU-miR-15a (Figure 4). Our results showed that 5 ng/mL and 10ng/mL TGFβ1 and 5-FU at 1mM and 50μM reduced the viability of PSCs. Importantly, 5-FU-miR-15a achieved the most potent inhibition of PSCs viability at nanomolar concentrations, while CTRL miR and miR-15a exert only a weak inhibitory effect. . Dose-response treatment of murine pancreatic stellate cells. Pancreatic stellate cells were treated with TGFβ1, 5-FU, CTRL miR, miR-15a, and 5-FU-miR-15a for six days, and cell viability was assessed using Cell-Titer Glo and compared to the control (medium). Data are represented as the mean ± SD; N = 4. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by One-Way ANOVA.
TGFβ1 is a proinflammatory factor that stimulates the activation of PSCs during the development and progression of chronic pancreatitis towards early pancreatic neoplasia [42][43][44]. Its role in the increase in α-SMA and the production of extracellular matrix has been well established in PSCs [19]. In addition, TGFβ1 induces multiple pathways and growth factors, including FGF and PDGF, that might result in an increase in proliferation. However, multiple studies show that TGFβ1 may exert no effect on proliferation or show inhibitory effects [45]. Thus, to assess the effect of 5-FU-miR-15a on PSCs proliferation in more detail, we performed a time course analysis by treating cells as a single agent and in the context of TGFβ1. Our results showed that TGFβ1, 5-FU, and 5-FU-miR-15a reduced the viability of PSCs at each tested time point, with the last treatment demonstrating the most significant reduction in cell viability ( Figure 5A-D) as compared to the controls. Simultaneously, treatment with miR-15a only slightly reduced the proliferation of PSCs. . Dose-response treatment of murine pancreatic stellate cells. Pancreatic stellate cells were treated with TGFβ1, 5-FU, CTRL miR, miR-15a, and 5-FU-miR-15a for six days, and cell viability was assessed using Cell-Titer Glo and compared to the control (medium). Data are represented as the mean ± SD; N = 4. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by One-Way ANOVA.
TGFβ1 is a proinflammatory factor that stimulates the activation of PSCs during the development and progression of chronic pancreatitis towards early pancreatic neoplasia [42][43][44]. Its role in the increase in α-SMA and the production of extracellular matrix has been well established in PSCs [19]. In addition, TGFβ1 induces multiple pathways and growth factors, including FGF and PDGF, that might result in an increase in proliferation. However, multiple studies show that TGFβ1 may exert no effect on proliferation or show inhibitory effects [45]. Thus, to assess the effect of 5-FU-miR-15a on PSCs proliferation in more detail, we performed a time course analysis by treating cells as a single agent and in the context of TGFβ1. Our results showed that TGFβ1, 5-FU, and 5-FU-miR-15a reduced the viability of PSCs at each tested time point, with the last treatment demonstrating the most significant reduction in cell viability ( Figure 5A-D) as compared to the controls. Simultaneously, treatment with miR-15a only slightly reduced the proliferation of PSCs. Notably, 5-FU-miR-15a in the context of TGFβ1 treatment manifested the most potent inhibitory effect while other treatments only slightly reduced PSCs proliferation ( Figure 5E-H). Together, these results demonstrate that 5-FU-miR-15a alone and in the context of TGFβ1 treatment showed the most significant reduction in PSCs proliferation. Notably, 5-FU-miR-15a in the context of TGFβ1 treatment manifested the most potent inhibitory effect while other treatments only slightly reduced PSCs proliferation ( Figure 5E-H). Together, these results demonstrate that 5-FU-miR-15a alone and in the context of TGFβ1 treatment showed the most significant reduction in PSCs proliferation. . Data are represented as the mean ± SD; N = 12 for a single treatment and N = 6 for a double treatment. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by One-Way ANOVA.

5-FU-miR-15a Significantly Reduced Migration of Murine Pancreatic Stellate Cells and Invasion of Pancreatic Cancer Cells In Vitro
In addition to their capability to proliferate during the development and progression of chronic pancreatitis, PSCs can migrate [46][47][48]. To investigate the impact of 5-FU-miR- . Data are represented as the mean ± SD; N = 12 for a single treatment and N = 6 for a double treatment. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by One-Way ANOVA.

5-FU-miR-15a Significantly Reduced Migration of Murine Pancreatic Stellate Cells and Invasion of Pancreatic Cancer Cells In Vitro
In addition to their capability to proliferate during the development and progression of chronic pancreatitis, PSCs can migrate [46][47][48]. To investigate the impact of 5-FU-miR-15a on the migration capacity of PSCs, we performed a "scratch" test. The results presented in Figures 6 and 7 show that 5-FU-miR-15a reduced the migration of PSCs most significantly as compared to controls when used as a single agent and in combination with TGFβ1 12 h and 22 h after the scratch. It has also been shown that PSCs drive the development and progression of pancreatic cancer. Thus, we performed an invasion assay to investigate whether treatment of PSCs with 5-FU-miR-15a reduced their capability to stimulate pancreatic cancer cell invasion. Conditioned media obtained from PSCs treated with TGFβ1, 5-FU, CTRL miR, miR-15a, and 5-FU-miR-15a alone and in combination with TGFβ1 were used as chemoattractants for pancreatic cancer cell invasion. As shown in Figure 8A,B, 5-FU-miR-15a alone and in combination with TGFβ1 most efficiently reduced the invasion of pancreatic cancer cells as compared to appropriate controls. 15a on the migration capacity of PSCs, we performed a "scratch" test. The results p sented in Figures 6 and 7 show that 5-FU-miR-15a reduced the migration of PSCs mo significantly as compared to controls when used as a single agent and in combination w TGFβ1 12 h and 22 h after the scratch. It has also been shown that PSCs drive the dev opment and progression of pancreatic cancer. Thus, we performed an invasion assay investigate whether treatment of PSCs with 5-FU-miR-15a reduced their capability stimulate pancreatic cancer cell invasion. Conditioned media obtained from PSCs treat with TGFβ1, 5-FU, CTRL miR, miR-15a, and 5-FU-miR-15a alone and in combination w TGFβ1 were used as chemoattractants for pancreatic cancer cell invasion. As shown Figure 8A,B, 5-FU-miR-15a alone and in combination with TGFβ1 most efficiently reduc the invasion of pancreatic cancer cells as compared to appropriate controls.

5-FU-miR-15a Significantly Reduced Inflammatory Markers in Pancreatic Stellate Cells
During the development of chronic pancreatitis, multiple signaling pathways are activated in PSCs [16,19,49]. In addition, miR-15a has been shown to impact the levels of inflammatory markers in various diseases [50][51][52][53]. Thus, we investigated the ability of 5-FU-miR-15a to alter expression levels of the components of these pathways. We treated PSCs with DMSO (vehicle) and 5 ng/mL TGFβ1, tested compounds (5-FU, CTRL miR, miR-15a, and 5-FU-miR-15a) at 50 nM concentration, and collected the cells for RNA and protein analysis after 24 h. We showed a slight increase in the expression levels of Yap1, Bcl-2, and Mmp9 and high induction of Il6 levels upon TGFβ1 stimulation ( Figure 9A-D). By itself, 5-FU at the tested concentration only marginally inhibited Yap1, Mmp9, and Il6. We have noticed that miR-15a and 5-FU-miR-15a can reduce mRNA levels of Yap1, Bcl-2, Mmp9, and Il6, with 5-FU-miR-15a having the most significant impact. Western blot analysis confirmed the inhibitory effect of 5-FU-miR-15a on YAP1 and BCL-2 by itself ( Figure  9A) and during co-treatment with TGFβ1 ( Figure 9B). . Pancreatic cancer cells were exposed to conditioned media obtained from pancreatic stellate cells treated with TGFβ1, and/or 5-FU, CTRL miR, miR-15a, and 5-FU-miR-15a.

The invasion was assessed 22 h post-treatment with (A) media obtained from pancreatic stellate cells after single agent treatment and (B) media obtained from pancreatic stellate cells co-treated with
TGFβ1. Data are represented as the mean ± SD, N = 8. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by one-way ANOVA.

5-FU-miR-15a Significantly Reduced Inflammatory Markers in Pancreatic Stellate Cells
During the development of chronic pancreatitis, multiple signaling pathways are activated in PSCs [16,19,49]. In addition, miR-15a has been shown to impact the levels of inflammatory markers in various diseases [50][51][52][53]. Thus, we investigated the ability of 5-FU-miR-15a to alter expression levels of the components of these pathways. We treated PSCs with DMSO (vehicle) and 5 ng/mL TGFβ1, tested compounds (5-FU, CTRL miR, miR-15a, and 5-FU-miR-15a) at 50 nM concentration, and collected the cells for RNA and protein analysis after 24 h. We showed a slight increase in the expression levels of Yap1, Bcl-2, and Mmp9 and high induction of Il6 levels upon TGFβ1 stimulation ( Figure 9A-D). By itself, 5-FU at the tested concentration only marginally inhibited Yap1, Mmp9, and Il6. We have noticed that miR-15a and 5-FU-miR-15a can reduce mRNA levels of Yap1, Bcl-2, Mmp9, and Il6, with 5-FU-miR-15a having the most significant impact. Western blot analysis confirmed the inhibitory effect of 5-FU-miR-15a on YAP1 and BCL-2 by itself ( Figure 9A) and during co-treatment with TGFβ1 ( Figure 9B).

5-FU-miR-15a Significantly Reduced Proliferation of Human Pancreatic Stellate Cells and Expression of YAP1 and BCL-2 In Vitro
We tested three commercially available PSCs of human origin (please see Materials and Methods Section) to investigate whether 5-FU-miR-15a has similar efficacy in inhibiting the proliferation of murine and human PSCs. Our results demonstrate that treatment with 5-FU-miR-15a most effectively reduces the proliferation of three human pancreatic stellate cells compared to appropriate controls ( Figure 10). Importantly, 5-FU-miR-15a exerts its inhibitory effects alone and with TGFβ1 co-treatment in all tested cell lines.

5-FU-miR-15a Significantly Reduced Proliferation of Human Pancreatic Stellate Cells and Expression of YAP1 and BCL-2 In Vitro
We tested three commercially available PSCs of human origin (please see Material and Methods section) to investigate whether 5-FU-miR-15a has similar efficacy in inhibiting the proliferation of murine and human PSCs. Our results demonstrate that treatment with 5-FU-miR-15a most effectively reduces the proliferation of three human pancreatic stellate cells compared to appropriate controls ( Figure 10). Importantly, 5-FU-miR-15a exerts its inhibitory effects alone and with TGFβ1 co-treatment in all tested cell lines. In addition, to determine the effect of 5-FU-miR-15a on the expression of YAP1 and BCL-2, we treated three hPSCs with DMSO (vehicle) and 5 ng/mL TGFβ1, tested compounds (5-FU, CTRL miR, miR-15a, and 5-FU-miR-15a) at 50 nM, and collected the cells for RNA and protein analysis after 24 h. RNA and protein analyses showed that 5-FU-miR-15a most significantly decreases the levels of YAP1 and BCL-2 as compared to the controls (Figure 11). In addition, to determine the effect of 5-FU-miR-15a on the expression of YAP1 and BCL-2, we treated three hPSCs with DMSO (vehicle) and 5 ng/mL TGFβ1, tested compounds (5-FU, CTRL miR, miR-15a, and 5-FU-miR-15a) at 50 nM, and collected the cells for RNA and protein analysis after 24 h. RNA and protein analyses showed that 5-FU-miR-15a most significantly decreases the levels of YAP1 and BCL-2 as compared to the controls ( Figure 11).

Discussion
Chronic pancreatitis is a fibroinflammatory disease that results in fibrosis and atrophy of the pancreas, and dysfunction of endocrine and exocrine activity of the organ [1,3,4,8,54]. It has been demonstrated that PSCs play a vital role in the development of fibrosis and its progression and, thus, impacts pancreatic acinar cells and immune cell activation [10,12,19,49,[55][56][57][58]. Chronic pancreatitis is a risk factor for pancreatic cancer, and mutations in the proto-oncogene KRAS are found in nearly all cases of pancreatic cancer [59][60][61]. Importantly, mutations of KRAS are often found in early neoplastic lesions resulting from chronic pancreatitis. Therefore, it has been suggested that elevated Kras levels in combination with a persistent inflammatory injury during chronic pancreatitis lead to early neoplasia and increase the likelihood of developing pancreatic cancer [62,63]. Currently, none of the limited treatments address the development and progression of fibrosis due to the activation of PSCs. Due to the limited availability of chronic pancreatitis specimens from human patients, we employed well-defined animal models. We showed that treatment with cerulein in Ptf1aCre ERTM and Ptf1aCre ERTM ;LSL-KRAS G12D mice showed downregulation of miR-15a and upregulation of two of its known targets: YAP1 and BCL-2 (Figures 1 and 2). We demonstrated that Ptf1aCre ERTM ;LSL-KRAS G12D mice treated with cerulein for four weeks have significantly increased fibrosis and that some cells positive for αSMA have proliferative status (Ki-67 + ) ( Figure 3). Our in vitro studies determined that 5-FU-miR-15a suppresses the activation of pancreatic stellate cells (PSCs), as shown by reduced viability, proliferation, and migration (Figures 4-10).
Specifically, we showed that 5-FU-miR-15a significantly inhibits the expression of Yap1, Bcl2, Il6, and Mmp9 alone and during treatment with TGFβ1 in murine PSCs (Figure 9). The latter is particularly significant as it shows that 5-FU-miR-15a abrogates the role of TGFβ1 during PSCs activation. RT-PCR analysis only showed a slight increase in Yap1 levels after TGFβ1 treatment at 24 h, and no increase at 48 h. In addition, we did not observe any substantial increase in the protein level of YAP1 upon TGFβ1. This is in agreement with previous data demonstrating that TGFβ1 has no impact on the status of YAP1. However, the crosstalk between YAP1/ TGFβ1 with YAP1 regulates the fibroinflammatory response of PSCs due to TGFβ1 stimulation [11]. Other studies showed that YAP-TEAD activation requires TGFβ1 signaling in cancer-associated fibroblasts and pancreatic stellate cell myofibroblasts [64,65]. Similarly, BCL-2 levels have been shown to not be affected or transiently affected by TGFβ1 signaling [66][67][68][69]. It is crucial that similar results regarding YAP1 and BCL2 reduction upon 5-FU-miR-15a treatment were obtained from murine and human PSCs ( Figure 11). Regarding Yap1 and Bcl2, our results are consistent with previous studies, showing the inhibitory effect of miR-15a and 5-FU-miR-15a on these factors' expression [29,31]. Importantly, we showed that condition media obtained from PSCs treated with 5-FU-miR-15a or TGFβ1, and 5-FU-miR-15a significantly inhibit the invasion of pancreatic cancer cells (Figure 8). These results, and previously published data regarding pancreatic cancer cells, show that treatment with 5-FU-miR-15a may simultaneously inhibit the proliferation and migration of pancreatic stellate cells and pancreatic cancer cells [29]. Further studies employing three-dimensional co-culture studies of pancreatic stellate cells and pancreatic cancer cells should shed light on the efficacy of 5-FU-miR-15a in inhibiting the proliferation and migration of both types of cells.
Furthermore, recent studies showed that multiple miRs (miR-301, miR-200c, miR-149, miR-139, miR-34b) can affect the activity of PSCs during chronic pancreatitis, and modulation of their levels leads to PSCs apoptosis [70][71][72][73][74]. Moreover, studies demonstrated that targeting PSCs with antifibrotic or anti-inflammatory compounds in a form of siRNA or small molecules could prevent chronic pancreatitis progression [75][76][77][78][79][80][81][82]. YAP1 has been shown to induce the activity of PSCs and stimulate pancreatic fibrosis during chronic pancreatitis [83]. Spanehl and colleagues showed that siRNA against Yap1 or verteporfin (YAP1 inhibitor) inhibits the proliferation and expression of fibrotic markers in activated rat PSCs [83]. HDAC inhibitors have been shown to increase the levels of miR-15 and miR-16, and lead to PSCs apoptosis and reduction in pancreatic fibrosis [84]. In vitro studies in activated rat PSCs showed that restoring the levels of miR-15b and miR-16 reduces Bcl2 and leads to apoptosis [27]. Notably, our results showed that 5-FU-miR-15a suppresses inflammatory and fibrotic markers (Mmp9 and Il6), thus providing a broader targeting pattern. In summary, our results demonstrate that ectopic delivery of 5-FU-miR-15a mimetics is a promising therapeutic approach for pancreatic fibrosis.

Animal Study
All studies and procedures involving animal subjects were approved by the Stony Brook University Institutional Animal Care and Use Committee (IACUC) and conducted strictly following the approved animal handling protocol. Ptf1aCre ERTM (Jackson Laboratory, Bar Harbor, Maine; Stock number 019378) and LSL-Kras G12D (Jackson Laboratory, Stock Number: 008179) mice have been described previously. All mice were maintained on a mixed background. Ptf1aCre ERTM ;LSL-Kras G12D mice were generated by crossing

Cell Lines
Murine pancreatic stellate cells were obtained from the laboratory of Dr. Means [85].

Cell Viability and Proliferation Assays
For cell viability experiments, PSCs were seeded at 10 3 cell density in 100 µL of a medium in a 96-well white microplate and treated with variable concentrations of TGFβ1, 5-FU, CTRL miR, miR-15a, and 5-FU-miR-15a. Cell viability was assessed six days after the treatments using Cell-Titer Glo (Promega, Madison, WI, USA, Cat. G7571) assay according to the manufacturer's protocol.
For cell proliferation experiments, PSCs were seeded at 2 × 10 5 cell density in 2 mL of a medium in a 6-well microplate and treated with 5-FU, CTRL miR, miR-15a, and 5-FU-miR-15a at 50 nM concentration alone or in combination with TGFβ1 at 5 ng/mL. The cells were collected at 24, 48, 96, and 144 h after the treatment and counted using a Z2 Cell Counter (Beckman Coulter, Inc., Brea, CA, USA).

Cell Migration
For cell migration experiments, PSCs were seeded at 2 × 10 5 cell density in 2 mL of a medium in a 6-well microplate and treated with DMSO, 5-FU, CTRL miR, miR-15a, and 5-FU-miR-15a at a 50 nM concentration alone and in combination with TGFβ1 at 5ng/mL for 24 h. Subsequently, a scratch was made using the tip of a 1000 µL pipette. Cell migration was evaluated at 12 and 22 h after the scratch using Nikon Eclipse Ti2 (Nikon Instruments, Inc., Melville, NY, USA) and analyzed using ImageJ Software [87].

Cell Invasion
PSCs were seeded at 2 × 10 5 cell density in 2 mL of a medium in a 6-well microplate and treated with DMSO, 5-FU, CTRL miR, miR-15a, and 5-FU-miR-15a alone at a 50 nM concentration and in combination with TGFβ1 at 5 ng/mL for 24 h. Pancreatic cancer cells were seeded in CultreCoat 96well Low BME plate (R&D Systems, Cat. 3481-096-K) on top of the transwell, and conditioned media obtained from PSCs treatments were added to the bottom of the chambers. The plate was incubated for 22 h at 37 • C in a CO 2 incubator. The measurements were done according to the manufacturer's protocol.

Quantification of Western Blot
Densitometry analysis was performed using ImageJ software [87].

Statistical Analysis
The analysis of in vitro experiments were performed using One-Way ANOVA or ttest with a value of p < 0.05 considered significant. This analysis was performed using GraphPad Prism version 9.4.1 for macOS (GraphPad Software, www.graphpad.com).

Conclusions
In summary, we showed that 5FU-modified miR-15a inhibits the expression of YAP1 and BCL-2 in murine and human PSCs in the in vitro setting. Importantly, we demonstrated that 5FU-modified miR-15a suppresses PSCs proliferation and migration, and inhibits the invasion of pancreatic cancer cells.